Microfluidic system with combined electrical and optical detection for high accuracy particle sorting and methods thereof

ABSTRACT

Disclosed herein is a system to detect and characterize individual particles and cells using at least either optic or electric detection as the particle or cell flows through a microfluidic channel. The system also provides for sorting particles and cells or isolating individual particles and cells.

RELATED APPLICATIONS

This invention claims the benefit of and priority to U.S. Ser. No.62/456,069, filed Feb. 7, 2017, the contents of which is herebyincorporated by reference in its entirety.

The present patent application incorporates by reference U.S. Pat. No.9,201,043, titled “Devices for Sorting a Particle in a Sample andMethods for Use Thereof,” International Patent Application PublicationNo. WO 2014/150928, titled “Devices for Sorting Cells in a Sample andMethods for Use Thereof,” International Patent Application PublicationNo. WO2013/052890, titled “Devices for Detecting a Particle in a Sampleand Methods for Use Thereof,” U.S. Provisional Application No.62/020,279, titled “Devices for Separating Analytes in a Sample,” U.S.Provisional Application No. 62/155,363 “Devices for Separating Analytesin a Sample and Methods for Use Thereof,” and U.S. ProvisionalApplication No. 62/346,934 “Metal Node-Pore Sensing,” in theirentireties.

TECHNICAL FIELD

The subject technology generally relates to detecting, characterizing,sorting, and/or isolating particles.

BACKGROUND

Existing live, single-cell isolation methods includefluorescence-activated cell sorting (FACS), magnetic-activated cellsorting (MACS), serial dilution, micromanipulation, and manual-picking.

FACS and MACS isolation techniques typically lead to the segregation ofmultiple cell groupings, rather than the isolation of a single cell.FACS and MACS are also ineffective at sorting and isolating single cellsfrom mixed or heterogeneous cell sample inputs. Such techniques aredifficult to optimize and are often restricted to core facilities, andconsecutive samples analyzed by the same fixed system pass through thesame valving, potentially leading to sample contamination.

Serial dilution involves diluting a sample containing cells of interestuntil there is a statistical likelihood that each aliquot of the dilutedsample contains exactly one cell. This technique is labor intensive andresults in a low isolation frequency. A high percentage of the targetcells of interest may not be present in the input sample, and serialdilution does not provide initial phenotypic information concerning thecells that are present in the input sample. Additionally, serialdilution results in a high variability and lack of reproducibility fromisolation to isolation.

Micromanipulation and manual-picking involve the physical selection andisolation of individual cells, but these methods are labor intensive andlow throughput.

SUMMARY

Disclosed herein are systems and methods for detecting, characterizing,sorting, and/or isolating particles of interest based on electricaland/or optical detection, and microfluidic flow.

Accordingly, in one aspect described herein is a particle sortercartridge that comprises a microfluidic chip including a sensing ordetector region and may further, optionally comprise a body defining aplurality of reservoirs and/or an interface plate configured to connectto the body and the microfluidic chip, where, upon detection of aparticle in the particle flow within the sensing or detector region, aflow of solution within the microfluidic chip is diverted by theapplication of a trigger flow to sort and/or isolate the detectedparticle from the particle flow.

In some embodiments, the microfluidic chip, the body, the optionalinterface plate, or a combination of these elements is disposable.

In some embodiments, the reservoirs defined by the cartridge bodyinclude a sample reservoir, a control fluid reservoir, and a triggerreservoir. In some embodiments, the trigger flow comprises a fluid flowfrom the trigger reservoir.

In some embodiments, the particle sorter cartridge further comprises anoptical detector, an electrical detector, or an optical detector and anelectrical detector, and the detector or detectors are operably coupledto the detector region of the microfluidic chip. In some embodiments,the detection of a particle in the detector region is based upon asignal generated by the electrical detector. In some embodiments, thedetection of a particle in the detector region is based upon a signalgenerated by the optical detector. In some embodiments comprising bothan electrical detector and an optical detector, the detection of aparticle in the detector region is based upon a signal generated by theelectrical detector, a signal generated by the optical detector, or asignal generated by the electrical detector and a signal generated bythe optical detector.

In some embodiments, the cartridge is the cartridge depicted in FIG. 2.

In another aspect provided herein a particle sorting system comprising abase plate and a cartridge assembly, where the base plate is configuredto receive and connect to the cartridge assembly, and the cartridgeassembly includes a microfluidic chip (including a sensing or detectorregion), and a body defining a plurality of reservoirs. The cartridgeassembly is configured such that, upon detection of a particle in aparticle flow within the detector region, a solution flow within themicrofluidic chip is diverted by application of a trigger flow to sortand/or isolate the detected particle from the particle flow.

In some embodiments, the microfluidic chip, the cartridge body, or boththe microfluidic chip and the cartridge body are disposable.

In some embodiments, the reservoirs defined by the cartridge bodyinclude a sample reservoir, a control fluid reservoir, and a triggerreservoir.

In some embodiments, the trigger flow comprises a fluid flow from thetrigger reservoir. In some embodiments, the trigger flow from thetrigger reservoir is gated by valving. In some embodiments, the triggerflow valving is external to the cartridge assembly.

In some embodiments, the cartridge assembly is further configured sothat, upon application of a trigger flow, a solution flow within themicrofluidic chip is diverted by valving of one or more carrier flowoutlets to sort and/or isolate the detected particle from the particleflow. In some embodiments, the carrier flow comprises fluid from thecontrol fluid reservoir. In some embodiments, the flow from the controlfluid reservoir is gated by valving. In some embodiments, the valving isexternal to the cartridge assembly.

In another aspect provided herein a particle sorting system is theparticle sorting system described in Example 8.

In some embodiments, a method for the active isolation of a particle isprovided, the method comprising loading a homogeneous or heterogeneoussample mixture or suspension of particles into the cartridge assembly,detecting a particle of interest by optical, electrical, or optical andelectrical signals generated by the particle of interest as it passesthrough the sensing or detector region of the microfluidic chip,actively sorting the particle of interest based upon the optical and/orelectrical signal or signals through software means that causes thesolution flow within the microfluidic chip to divert by the applicationof a trigger flow, and depositing a droplet comprising theparticle-of-interest into a collection receptacle. In some embodiments,the method for the active isolation of a particle comprises the particlesorting system described in Example 8.

In some embodiments, the detecting step provides a method to measure thevelocity of the particle traversing the sensing or detector region. Insome embodiments, the method allows for precise triggering of opticaldetection for quantitative and reproducible measurements for particlecharacterization as well as minimizing the time the particle is exposedto an optical signal. This method can further comprise a step ofincreasing the velocity of the particle of interest as it passes throughthe sensing or detecting region such that the total time the particle isexposed to an optical signal is less than 50, 60, 70, 80, 90, 100, 110,120, 130, 140, or 150 milliseconds. In some embodiments, the methodfurther comprises a step of increasing the velocity of the particle ofinterest as it passes through the sensing or detecting region such thatthe total time the particle is exposed to an optical signal is less than100 milliseconds.

In one aspect provided herein is a method for the active isolation of aparticle of interest comprising flowing a mixture of particles through amicrochannel comprising a detector or sensing region, detecting aparticle of interest by optical and/or electrical signals generated bythe particle of interest as it passes through the sensing or detectorregion of the microchannel, upon the detection of a particle of interestin the detector or sensing region, diverting the flow of the particlesolution by the application of another flow, thereby actively sortingthe particle, and depositing a droplet comprising the particle ofinterest into a collection receptacle.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the subject technology and many of itsadvantages will be understood by reference to the following detaileddescription when considered in connection with the following drawings,which are presented for the purpose of illustration only and are notintended to be limiting.

FIG. 1 is a diagram showing a broadly applicable system platform andbasic workflow, according to some embodiments.

FIGS. 2A-2I illustrate a body of the cartridge, according to someembodiments.

FIGS. 3A-3F illustrate an interface plate, according to someembodiments.

FIGS. 4A-4B illustrate a microfluidic chip mold, according to someembodiments.

FIGS. 5A-5C depict the assembly of an exemplary microfluidic chip with amicrofluidic chip mold, according to some embodiments.

FIGS. 6A-6G illustrate an assembled microfluidic chip and microfluidicchip mold, with detection and sorting regions, according to someembodiments.

FIGS. 7A-7I and 8A-8I illustrate a cartridge assembly, including acombination of the components of FIGS. 2A-6G

FIGS. 9A-9N illustrate an exemplary base plate of an associated hardwareplatform, according to some embodiments.

FIGS. 10A-10B illustrate a cartridge mated with a base plate of ahardware platform, according to some embodiments.

FIGS. 11A-11L illustrate a lid with a sealing surface, according to someembodiments.

FIGS. 12A-12L illustrate a pressure regulator manifold, according tosome embodiments.

FIG. 13 depicts an exemplary subsystem layout, according to someembodiments.

FIGS. 14A-14C are a schematic representation of the fluid paths in theexemplary subsystem depicted in FIG. 13.

FIG. 15 is an embodiment of an LED-based fluorescent detection system,according to some embodiments.

FIGS. 16A-16D depict an example of an overall system assembly, accordingto some embodiments.

FIG. 17 illustrates an exemplary scheme for particle detection and/orsensing and isolation, according to some embodiments.

FIGS. 18A-18C are experimental data plots showing cell sorting ofcirculating tumor cells from a blood sample, according to an exampleimplementation.

FIGS. 19A-19C are micrographs illustrating cell isolation, according toan example implementation.

FIG. 20 is an experimental data plot showing the viability of cellssorted according to an exemplary implementation is maintained relativeto unsorted cells.

FIG. 21 is a micrograph illustrating that fluidic sorting according toan example implementation is independent of particle size. MCF7 cellclusters isolated using the platform are shown in circled regions.

FIGS. 22A-22C are fluorescent micrographs illustrating cell isolation,according to an example implementation utilizing fluorescence basedselection.

DETAILED DESCRIPTION

Systems and cartridges described herein advantageously providefunctionality for simultaneously performing two or more of the functionsbelow:

-   -   Detect, sort, and isolate with sheathless operation;    -   Accelerate drop creation and dispensing with the carrier line        (not needing to use the sample line) to improve functional        sorting frequency. To reach a minimum volume needed for        dispensing/dropping, sufficient fluid must exit the collection        outlet, and designs described herein make this possible without        flowing more sample into the collection outlet than minimally        desired. Drop creation and dispensation timings can also be        tuned without needing to change the sample flow rate;    -   Minimize fluid into the collection outlet leading to drop        creation when no particle is detected. Because of the carrier        waste pathway and external valving, carrier flow can flow        primarily through the carrier waste outlet when no particle of        interest is detected, drastically reducing the collection of        droplets that do not contain particles of interest;    -   Selectively diverting particles from the collection outlet, even        downstream of detection, such that drops exiting the collection        outlet can be ensured to only contain the desired number of        particles (e.g., one particle, when implemented for single        particle dispensing); and    -   Control particle direction without valving the sample (i.e.,        particles from one test never contact surfaces that particles        from a previous test contacted). By virtue of how the fluid is        routed according to designs described herein, only the carrier        and trigger fluid paths ever leave the cartridge and are valved        externally (for example as shown in FIGS. 14A-14C, illustrating        fluid paths).

System Overview

FIG. 1 shows the broadly applicable system platform and basic workflow.An input sample can be essentially any sample, including pre-enrichedcell samples, whole blood, plasma, dissociated tissue, cultured celllines, transfected cells, urine, or agricultural, forensic, or generalbiological samples, as well as non-biological samples such as colloidalsolutions. The sample is loaded into a cartridge, which is then insertedinto a hardware platform containing a detection module and amicrofluidic module for the collection of desired particles of interestfrom the sample. For purposes of the present disclosure, “particles” canrefer to cells, cellular vehicles, virus, DNA, RNA, polymers, such aspolystyrene beads, latex beads, colloids (e.g., metal colloids),magnetic particles, dielectric particles, crystals (e.g., micro-crystalsor nano-crystals), bioparticles such as pores, pollen, cellularocclusions, precipitates, intracellular crystals, biological molecules,including viruses, peptides, antibodies, diabodies, etc., fragmentantigen-binding (Fab) fragments, binding proteins, phosphorylatedproteins, aptamers, epitopes, polysaccharides, polypeptides, proteins,lipids, peptidoglycans, phospholipids, sugars, glycoproteins, sugarchains, nucleic acids, ribosomes, and any other cellular components,particulates, fibers, impurities, contaminants, and synthetic particleshaving a size range of 0.01 nm to 100 mm. For example, single(individual) particles can be collected and each deposited intocorresponding separate wells of a microtiter plate through theintegration of the microfluidic module and robotic control of themicrotiter plate.

In some embodiments, the workflow for the instant technology cancomprise loading a fluid sample and buffers into the cartridge, placingthe cartridge into the base plate of the hardware platform, providingair pressure to the loaded fluids to produce a flow, sensing anddetecting particles as they pass through the sensing region or regionsof the microfluidic chip, and sorting and isolating the detectedparticles of interest.

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

“Particle,” as used herein, refers to any object that can becharacterized, detected, sorted, and/or isolated. Particles include butare not limited to DNA, RNA, polymers, such as polystyrene beads, latexbeads, colloids (e.g., metal colloids), magnetic particles, dielectricparticles, crystals (e.g., micro-crystals or nano-crystals),bioparticles such as pores, pollen, cellular occlusions, precipitates,intracellular crystals, biological molecules, including viruses,peptides, antibodies, diabodies, etc., Fab fragments, binding proteins,phosphorylated proteins, aptamers, epitopes, polysaccharides,polypeptides, proteins, lipids, peptidoglycans, phospholipids, sugars,glycoproteins, sugar chains, nucleic acids, ribosomes, and any othercellular components, particulates, fibers, impurities, contaminants andsynthetic particles having a size range of 0.01 nm to 100 mm.

“Cell of interest” or “target cell,” as used herein, refers to abiological particle that it is desirable to detect, characterize, sort,or isolate. In some embodiments, the cell of interest or target cellwill be a functional unit of an organism, tissue, or a single-cellorganism. In some embodiments, a cell of interest or target cell is partof a class or category of functional units of an organism, tissue, orsingle-cell organisms. Cells of interest or target cells may becancerous or precancerous cells or they may be normal or not diseasedcells.

“Cartridge,” as used herein, refers to a container that comprises atleast a cartridge body, a chip substrate, and a chip mold. In someembodiments, the cartridge also comprises an interface plate. In someembodiments, the cartridge is a “disposable cartridge,” wherein thecartridge body, chip substrate, chip mold, another cartridge bodycomponent, or any combination of cartridge components is disposable.

The cartridge body of a cartridge comprises at least one samplereservoir and at least one control fluid reservoir. The chip moldcomprises microchannels that can carry a fluid or fluids from thecartridge body of a cartridge. The chip substrate and chip mold alsocomprises detection and sorting regions. In some embodiments, thecartridge body is connected to the chip substrate and mold through aninterface plate.

The term “sample reservoir” (or “sample cavity”) as used herein refersto a reservoir of the cartridge body that can contain a fluid, solution,or mixture. In preferred embodiments, these fluids, solutions, ormixtures comprising cells or particles of interest includingheterogeneous mixtures of cells or particles. The volume of fluid thatcan be contained in a sample reservoir can be as little as 1 microliter.

“Control fluid reservoir” (or “control reservoir” or “control cavity”)as used herein refers to a reservoir of the cartridge body that cancontain a fluid, solution, or mixture. This fluid, solution, or mixturemay be a buffer or buffer mixture, a cellular medium or cellular mediamixture, or any reagent or reagents. In some embodiments, the controlfluid reservoir is a “carrier” reservoir that provides a flow stream toadvance particles from the sample fluid towards the collection outlet.In some embodiments, the control fluid reservoir is a “trigger”reservoir that selectively directs detected and/or characterizedparticles from the sample fluid. As described herein, in someembodiments, a plurality of control fluid reservoirs (e.g., including atrigger reservoir and a carrier reservoir) are used in a singleimplementation.

The term “light pipe,” as used herein, refers to a hole, aperture, bore,void, or other passageway for light. In some embodiments, the light pipeis an open cylinder made of a material that prevents the passage oflight except for light from the light source (positioned at the top ofthe pipe) and light that is emitted back from particles, includinglabeled particles, in the sensing or detector region of the microfluidicchip (positioned beneath the bottom of the pipe). In some embodiments,the light pipe can be made of open space and/or transparent material notlimited to glass, PMMA, cyclic-olefin-copolymer, or any similarmaterials known to practitioners of the art. In preferred embodiments,the light pipe allows passage through the cartridge body and interfaceplate to the microchip substrate and mold. The light may be anywavelength or mixture of wavelengths. In some embodiments, the lightsource can provide specific excitation wavelengths or mixture ofwavelengths using light filters as is known in the art. The light thattravels through the light pipe may be excitation light, emission light,or both.

“Interface plate,” as used herein, refers to an object that connects thecartridge body to the microfluidic chip. The interface plate providespassage for fluids in the reservoirs of the cartridge body to themicrochannels of the microfluidic chip. In some embodiments, theinterface plate is the location of the microphase-macrophase transitionor transitions, or interface or interfaces. The interface plate mayprovide the connection between the cartridge body and the microfluidicchip in any manner, including fluid passages, valving, or other means.

The term “microfluidic chip,” as used herein, refers to an objectcomprising a microfluidic substrate and a microfluidic mold. Themicrofluidic chip comprises passages or microchannels for the fluidsloaded into the reservoir or reservoirs of the cartridge body. One ormore of these microchannels can comprise one or more sensing regions.One or more of these microchannels can comprise one or more sortingregions.

The term “sensing region,” as used herein, refers to a portion of amicrofluidic chip that comprises a portion of a microchannel thatproduces a detectable signal or signal change as a particle of interesttravels through it. In preferred embodiments, the detectable signal orsignal change occurs with respect to individual particles as they passserially, one-by-one, through the sensing region. The detectable signalor signal change can be of any type or combination of types. Particulartypes of signal or signal change, dependent upon the embodiment, caninclude optic or fluorescent and electrical signals.

The term “fluorescent detection,” as used herein, refers to sensing asignal or change in signal, the signal being a light wavelength orwavelengths, such as ultraviolet light or visible light. The signalbeing sensed can be produced or altered by a particle emitting a lightwavelength (e.g., an emission signal) in response to exposure to anexcitation light.

The term “electrical detection,” as used herein, refers to sensing asignal or change in signal, the signal being impedance, resistance, oranother characteristic of current. The signal being sensed can beproduced or altered by a particle crossing the path of an electricalcurrent. In some embodiments, electrical detection includes use of theCoulter Principle.

“Node pore sensing” (NPS), as used herein, refers to screening singleparticles, such as cells, by electrical detection as they pass throughone or more nodes. In some embodiments, NPS comprises one or moresurface functionalized regions with a retarding agent. Determining thetime a single particle is retarded provides characteristic informationconcerning that particle. References relating to NPS, which are herebyspecifically incorporated by reference, are U.S. Pat. No. 9,201,043 andBalakrishnan et al., (2015), Anal. Chem. 87 (5), 2988-2995.

“Sensitivity of detection,” as used herein, refers to the ability todetect and/or characterize a particle in the sensing region of theinstant technology. Sensitivity of detection can include, but is notlimited to, sensing electric and/or optic signals, or changes in suchsignals.

“Dynamic range,” as used herein, refers to the ability to detect signalsand changes in signals resulting from a particle in the sensing regionof the instant technology. The dynamic range includes both large-scalesignal detection (e.g., a binary no signal/signal system) andsmall-scale signal detection (e.g., differentiating between two similarbut distinct signals).

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

System Elements

In some embodiments, a system comprises a hardware platform, amicrofluidic cartridge, and one or more collection devices. The hardwareplatform provides the pneumatics, electronics, detection equipment(e.g., a photodiode light source or an LED light source and opticdetector, and/or a detector for electrical signals caused by passage ofa particle through the sensing region), and other elements of thesystem. For example, the hardware platform can include a housing,pneumatics module, collection module, electronics module, and adetection and sorting module. The housing can be used to support andalign each of the components of the instrument including the pneumatics,detection, sorting, and collection modules (FIGS. 9A-9N, FIGS. 10A-B,and FIGS. 16A-D). The design of the embodiment shown in FIG. 13 enablessingle-axis alignment as all contacts and/or interfaces occur in thesame axis. The pneumatics module can comprise three pressure sources—asample, a trigger, and a carrier, which feed into the different cavitiesof the microfluidic cartridge. The collection module houses the outputcontainer, which can include a plurality of collection chambers (e.g., 1for a single collection tube, 96 for a 96-well microtiter plate, 384 fora 384-well microtiter plate, etc.) depending on the desired application.FIG. 13 depicts an embodiment where the collection module comprises a96-well microtiter plate. The collection module can be moved (i.e.,actuated or “translated”) to collect drops into collection chambers fromthe cartridge as desirable.

The electronics module can include the electric subsystem of thehardware platform and the stage subsystem (e.g., well plate driver) ofthe hardware platform.

The detection and sorting module can comprise a detection region whicheither detects or determines a characteristic of a particle in thesample fluid and a sorting region which switches the particle into acollection flow stream. An exemplary implementation of the detection andsorting module is depicted in FIG. 17.

The preferred microfluidic cartridge is disposable. A disposablecartridge ensures that an individual sample does not come into contactwith a previous sample or interface with any surfaces on the cartridgeor instrument that a previous sample came into contact with. Themicrofluidic disposable cartridge comprises at least three stages ofarchitecture: (1) the body, (2) the microfluidic substrate, (3) and themicrofluidic chip mold. In some embodiments, a fourth stage, theinterface plate, is also included.

The body of the disposable cartridge can include a sample reservoir anda control fluid reservoir or a plurality of control fluid reservoirs.

The hardware platform enables the various modules of the system to actin concert.

The microfluidic cartridge comprises a cartridge body, a microfluidicchip substrate, and a microfluidic chip mold. The cartridge bodycomprises at least a sample reservoir and a control reservoir. Thesereservoirs are connected through fluid passages to the microfluidic chipsubstrate and the microfluidic chip mold. The microfluidic chipsubstrate and the microfluidic chip mold are bonded to form amicrofluidic chip. In some embodiments, the, microfluidic chip substrateand the microfluidic chip mold are bonded through one of, or acombination of, heat, lamination, and solvent, though the substrate andthe mold can be bonded by any appropriate method. The chip comprises atleast a microfluidic channel that enters a sensing region, a sensingregion, and a microfluidic channel that exits the sensing region thathas at least one sorting region to direct the flow of sample solutionfor single particle collection or isolation. In some embodiments, theconnection between the cartridge body and the microfluidic chipsubstrate and mold is provided by an interface plate comprising fluidpassages or “channels.” During use, fluid is routed through theinterface plate channels, enters the base plate (which houses valves),and then returns to the interface plate. The collection device providesa receptacle that can receive sample output in the form of sorted orisolated droplets from the microfluidic cartridge.

FIGS. 2A-6G show an embodiment of a cartridge assembly architecture. Thecartridge assembly can be described to have four components: a cartridgebody, an interface plate, a chip substrate, and a chip mold. One or moreof these components can be disposable.

FIGS. 2A-2I show a body of the cartridge. The cartridge body 200 has atleast a sample reservoir and a control fluid reservoir (each of whichmay also be referred to as a “cavity”). In the embodiment shown in theFIGS. 2A-2I, there are two control fluid reservoirs 202A, 202B, or“buffer reservoirs,” and one sample reservoir 204. One of the depictedcontrol fluid reservoirs 202 acts as a “carrier” reservoir that providesa flow stream to advance the particles to the collection outlet. Theother depicted control fluid reservoir acts as a “trigger” reservoir toselectively direct detected and/or characterized particles. In someembodiments, flow streams from the carrier and/or trigger reservoirs aredetermined in response to the flow stream provided by the samplereservoir. Any one or any combination of the reservoirs may comprise areservoir floor that possesses an incline, slope, or angle such thatliquids placed in the reservoir will continuously accumulate proximateto the reservoir's fluid inlet.

In some embodiments, the flow stream provided by the carrier reservoirhas a constant rate. In some embodiments, the flow stream provided bythe carrier reservoir is uninterrupted. In some embodiments, the flowstream provided by the trigger reservoir is provided as a pulse ortransient flow of solution.

In FIGS. 2A-2I, the sample reservoir 204 can function for its intendedpurpose with as little as 1 microliter of solution up to 10 millilitersof solution. There are no limits to the size of the sample reservoir204, which may contain any volume of sample solution. In someembodiments, the sample reservoir 204 may contain more than 1milliliter, 10 milliliters, 15 milliliters, 20 milliliters, 30milliliters, 50 milliliters, 100 milliliters, or 1 liter. The twocontrol fluid reservoirs 202A, 202B can function with as little as 1microliter of solution up to 10 or more milliliters of solution. The twocontrol fluid reservoirs can function with as little as 10 microlitersor less of solution up to 100 milliliters or more of solution. The twocontrol fluid reservoirs may be loaded with any appropriate solutions.In some embodiments, these solutions comprise a buffer or buffers, areagent or reagents, a medium or media, or any combination of buffers,reagents, and media.

As depicted in FIGS. 2A-2I (by way of example only), the cartridge body200 has overall dimensions of 54 mm×46 mm×32.4 mm.

The cartridge body 200 can comprise an inert material, such as acrylic,polycarbonate, cyclic olefin polymer/co-polymer (COP, COC),acrylonitrile butadiene styrene (ABS), polystyrene, or other suitablematerial. In one embodiment, the cartridge body 200 is machined out ofacrylic polymer. The cartridge body 200 can be formed via machining orinjection molding.

The body stage (i.e., one of the at least three stages of themicrofluidic cartridge) may have any overall dimensions appropriate forits use. In some embodiments, appropriate dimensions may be determinedin response to the volume or volumes of the liquid or liquids to beloaded into the sample reservoir or the control fluid reservoir orreservoirs of the body stage. In some embodiments, the dimensions of thebody stage will be determined in response to the dimensions of otherstages of architecture in the microfluidic cartridge. In someembodiments, the dimensions of the body stage may be determined by thehardware platform into which the cartridge is to be loaded.

To the bottom of the cartridge body 200 (an exemplary bottom view isshown in FIGS. 2A-2C), an interface plate can be attached. Fluid inletpassages (see, e.g., aperture 206) connect the sample reservoir andcontrol fluid reservoirs through the interface plate to the microfluidicchip. The diameter of the apertures configured to interface with fluidinlet passages (i.e., the smaller apertures—206) in FIG. 2 are 1/32inches, however the diameter of the apertures can be any size that issuitable for a given implementation. In some embodiments, one or more ofthe reservoirs' apertures have diameters unequal to one or morediameters of the other reservoirs' apertures. The fluid inlet passagesmay have any appropriate dimension to connect the sample and controlfluid reservoirs to the microfluidic chip. For example, the diameter ofthe fluid inlet entry in the interface plate (connecting to the bodystage) may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, orgreater than 1 millimeter. One or more of the apertures (e.g., aperture208) can serve as a light bore for optical detection, rather than forfluid transfer. In some embodiments, there is one aperture at the bottomof each reservoir/cavity of the cartridge body. A cavity of the samplereservoir 204 can be larger than the cavities of the control fluidreservoirs 202A, 202B.

In some embodiments, a light pipe core can be positioned through thecenter of the cartridge body and the interface plate. In suchembodiments, when the cartridge is placed into the base plate of thehardware platform, a fluorescent element (both emission element anddetection element) can be positioned above the light pipe core. Emissionand detection signals can travel through the light pipe core relative tothe sensing region in the microfluidic chip (see FIG. 15). In otherembodiments, there is only an electrical detection module and thereforethe light pipe core is optional or absent.

FIGS. 3A-3F show an exemplary interface plate. The interface plate 310contains fluid channels 312 configured to connect the sample and controlreservoirs of the cartridge body (e.g., the cartridge body 200 of FIGS.2A-2I) to the microfluidic chip. In some embodiments, valves andpressure regulators control the flow rate of solution from the sampleand control fluid reservoirs through the fluid channels 312 in theinterface plate 310 and then to fluid vias that connect the fluidchannels in the interface plate 310 to the microfluidic chip. Theinterface plate may be produced by any appropriate technique and in anyappropriate matter. In one embodiment the bodies are machined out ofpolycarbonate polymers. In another embodiment the body is formed throughinjection molding.

In some embodiments, the interface plate includes one or more of: fluidchannels that are configured to connect the sample and controlreservoirs in the body stage to the microfluidic chip, fluid channelsconnecting the trigger reservoir to the base-plate external valve andconnecting the base-plate external valve to the microfluidic chip, fluidchannels connecting the carrier waste outlet to an external valve andfrom the external valve to a waste outlet, and fluid channels connectingthe microfluidic chip to dispensing nozzles or outlets exiting thecartridge. In embodiments without an interface plate, these connectionsare provided directly between the appropriate stages of the microfluidiccartridge.

In some embodiments, the interface plate is adhered to the bottom of thebody stage. This is accomplished in certain embodiments by a pressuresensitive adhesive, glue, thermal bonding, ultrasonic welding, or othersuitable methods.

In some embodiments, the dispensing nozzles include a nozzle vibrationcavity that is controlled by software, where the control of vibrationcan modulate the flow rate of solution. In some embodiments, the nozzlevibration is provided by a piezoelectric device. In some embodiments,the interface plate has a plurality of nozzles that allow for buffer orsample to exit the cartridge. In some embodiments, the interface platehas one nozzle for collection and/or dispensing liquid. In someembodiments, one or more of the nozzles may be replaced with a hole thatfeeds into the base plate for routing liquids.

O-ring grooves can be included, to allow for sealed fluidic contact withpneumatic valves, for contact between the cartridge and the lid, or forcontact between the base plate and the cartridge.

FIG. 3F shows how the architecture of the interface plate 310 is alignedwith the architecture of a microfluidic chip. Ridges 316 for alignmentand bonding ensure proper attachment of the interface plate 310 with themicrofluidic chip.

The overall dimensions of the interface plate 310 as depicted in FIGS.3A-3F are 54 mm×46 mm×4.3 mm, however the interface plate may have anyoverall dimensions appropriate for its use. For example, in someembodiments, the dimensions of the interface plate will be determined inresponse to the dimensions of other stages of architecture in themicrofluidic cartridge. In some embodiments, the dimensions of theinterface stage may be determined by the hardware platform into whichthe cartridge is to be loaded.

FIGS. 4A-4B show an exemplary microfluidic chip mold 418, with labelledconnection points: sample collection, sample waste, carrier waste,carrier inlet, trigger inlet, and sample inlet.

FIGS. 5A-5C depict the assembly of an exemplary microfluidic chip 520with a microfluidic chip mold 518, which may be similar to microfluidicchip mold of FIGS. 4A-4B. The microfluidic chip 520 includes a pluralityof microfluidic channels 522 defined therein. The microfluidic substrateand mold can comprise a bioinert material that does not producecytotoxic events in cells that traverse the microfluidic channels. In apreferred embodiment, the microfluidic substrate and mold aretransparent to allow for optical imaging, fluorescent detection, and/orother optic detection and characterization. The microfluidic chip andmold can comprise any appropriate materials including acrylics, cyclinolefin polymers (COP) or copolymers (COC), and polycarbonate.

The microfluidic substrate comprises patterned electrodes that provideelectrical contact for sensing and detecting particles in a sensingregion. The electrodes may be made of any appropriate conductivematerial. In one embodiment, the electrodes comprise a copper adhesionlayer and a platinum top layer. The patterned electrodes may bepresented in any pattern so long as they provide contacts for sensingcurrent changes across a sensing region of the microfluidic chip mold.In some embodiments, there may be two or more electrodes. For instance,the detector may be a two-point (e.g., two-electrode) detector, athree-point (e.g., three-electrode) detector, a four-point (e.g.,four-electrode) detector, etc. (See U.S. Pat. No. 9,201,043, which ishereby incorporated-by-reference.)

The mold can comprise at least one microfluidic channel configured to beconnected to the reservoirs in the cartridge body through the fluid viasin the faceplate.

The microfluidic substrate and mold may be made of any appropriatesubstance and by any appropriate method. In some embodiments, either orboth may be injection molded. In some embodiments, either or both may bemade of a rigid material such as plastic. The thickness of themicrofluidic chip may be any appropriate thickness, including about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 millimeter, or greater than1 millimeter. The thickness of the microfluidic mold may be anyappropriate thickness, including about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, or greater than 1 millimeter.

The microfluidic substrate and mold can comprise at least: (1) amicrofluidic channel that enters the sensing region, (2) the sensingregion, and (3) a microfluidic channel that exits the sensing regionthat has at least one sorting region comprising a trigger that candirect the flow of solution into a carrier microfluidic channel forsingle particle collection/isolation. As such, according to certainembodiments described herein, cell sorting is performed actively (e.g.,via fluidic forces), rather than passively (e.g., via channel dimensionsand/or composition). In some embodiments, the carrier channel is splitinto a plurality of microfluidic channels, each connecting the carrierchannel to the sample collection outlet or to the carrier waste outlet.In some embodiments, a microfluidic channel connecting the carrierchannel to the sample collection outlet or to the carrier waste outletis externally valved through a connection with the interface and baseplate. In some embodiments, such microfluidic channel valving improvescollection drop volume creation by minimizing drop creation when noparticle of interest is sorted while maximizing drop creation when aparticle of interest is sorted.

The microfluidic substrate and mold comprise one or more inlets that areconnected to the reservoirs in the body through the fluid vias in thefaceplate. The sample reservoir is connected to at least one inlet inthe microfluidic substrate and mold. Other inlets may be connected tothe control buffer reservoirs.

A microfluidic channel runs from the sample inlet to a sensing region inthe microfluidic chip. When a flow is provided, the liquid in the samplereservoir flows into the sample inlet and then enters the sensingregion. In preferred embodiments, only fluids from the sample reservoirwill flow through a sample inlet into the sensing region.

The sensing region can detect a characteristic and/or the presence ofsingle cells and particles ranging in size from sub-micron to micronrange or larger. In some embodiments, detection occurs using the Coulterprinciple. A cell or particle passes through a conduit in the sensingregion. In some embodiments, a sensing region comprises a conduitproximal to one or more nodes. In such embodiments, the diameter of theconduit is smaller than the region or regions (node or nodes) of themicrofluidic channel that enters and exits the sensing region such thata particle can cause electrical impedance/resistance when it enters theconduit. In other embodiments, the sensing region comprises no nodes. Inother embodiments, the sensing regions comprises a plurality of nodesproximal to a plurality of conduits with smaller diameters. In suchembodiments, there may be two, three, four, five, or more than fivenodes. In some embodiments, a sensing region comprises a plurality ofconduits, each conduit having an entrance and an exit proximate to anode. In some embodiments, a sensing region comprises a plurality ofconduits, wherein less than every conduit entrance and/or exit isproximate to a node. In some embodiments, the sensing region can haveone of the designs as described in U.S. Pat. No. 9,201 043, which isincorporated-by-reference. In other embodiments, the conduit has morethan 2 nodes such that there are multiple events of electrical impedanceand resistance.

In a preferred embodiment, the system is for single cell detection,sorting, and isolation. In this case, the diameter and height of theconduit is at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,or 150 micrometers. The conduit must be large enough for a cell to passthrough the conduit, but also small enough for a single cell or particleto cause electrical impedance/resistance of the current flowing betweena pair of nodes of the conduit.

The range of cell diameters that can be detected by the presenttechnology is broad and encompasses many important cell diameters in thefield. For example, mature female eggs are among the largest cell typeswith about a 120 micrometer diameter. In terms of average volume(micrometer cubed), a red blood cell is 100, lymphocyte is 130,neutrophil 300, fibroblast 2,000, HeLa is 3,000, osteoblast 4,000, andan oocyte is 4,000,000.

It is envisioned that the technology may be used to detect andcharacterize particles smaller than a cell. An appropriately sizedconduit may be used to detect and characterize any sized particles. Suchparticles may include bioparticles such as proteins, peptides, aminoacids, lipids, fats, phospholipids, sugars, glycoproteins, sugar chains,nucleic acids, ribosomes, DNA, RNA, organelles, ribosomes, cellularvehicles, viruses, pores, pollen, cellular occlusions, precipitates,intracellular crystals, biological molecules, including viruses,antibodies, diabodies, etc., Fab fragments, binding proteins,phosphorylated proteins, aptamers, epitopes, polysaccharides,polypeptides, peptidoglycans, polymers, such as polystyrene beads, latexbeads, colloids (e.g., metal colloids), magnetic particles, dielectricparticles, crystals (e.g., micro-crystals or nano-crystals), and anyother cellular components, particulates, fibers, impurities,contaminants and synthetic particles having a size range of 0.01 nm to100 mm.

The conduit in the sensing region has an effective electrical impedanceor resistance that is changed with the passage of each cell or particletherethrough.

Nodes for electrical detection provide for information ascertained bythe Coulter Principle. The nodes are provided an electrical current bythe patterned electrodes of the microfluidic chip, and other patternedelectrodes provide sensing the current through the electrical detectionnodes. In some embodiments, the patterned electrodes comprise afour-point electrode or four-point measurement comprising two inner andtwo outer electrodes. In certain embodiments, the outer electrodesprovide a current (e.g., a constant current) into the conduit, and theinner electrodes detect changes in the electrical properties of theconduit (e.g., voltage). In other embodiments, the inner electrodesprovide a current into the conduit, and the outer electrodes detectchanges in the electrical properties of the conduit. In someembodiments, the outer electrodes provide a current to the conduit, andthe inner electrodes regulate the voltage provided to the conduit butpass no current.

Optical and electrical sensing locations within a sensing region can bedistributed in any appropriate manner. The optical sensing location orlocations in the sensing region are aligned with the light pipe bores inthe body and the faceplate such that it provides a “window ofobservation” for fluorescent detection.

The electrical sensing location or locations may be anywhere within thesensing region. In some embodiments, an electrical sensing region mayoverlap with an optical sensing location. In some embodiments, anelectrical sensing location may reside between the sample inlet and thefirst optical sensing location. In some embodiments, an electricalsensing location may reside between the sorting region and the lastoptical sensing location. In some embodiments, an electrical sensinglocation may reside between two optical sensing locations.

In a preferred embodiment, the detection or sensing region has bothoptical and electrical sensing capability.

In one embodiment, the detection region has a segmented conduit thatcomprises one or more nodes and two or more sections. Fluorescent meanscan provide the optical sensing capability. A preferred embodiment offluorescent means is shown in FIG. 15.

Simultaneous detection and characterization by electrical and opticalsignals provides the electrical signal or change in signal, the opticalsignal or change in signal, and a method to measure the velocity of theparticle traversing the signaling site, increasing the accuracy ofoptical measurements. In some embodiments, this increased accuracyallows for each particle to be exposed to an excitation wavelength forless time. Less exposure to the excitation wavelength may reducecytotoxic effects and photobleaching to particles (e.g., cells) ofinterest, boosting their viability and increasing ultimate yield.

A microfluidic channel exits out of the sensing region and connects toone or more sorting regions.

A sorting region comprises a microfluidic channel originating from thesample microfluidic channel and a branch point that results in two ormore separate microfluidic channels.

In some embodiments, one or more of the microfluidic channels exiting asorting region connect the sorting region to an outlet in themicrofluidic substrate and mold. The outlet or outlets may be used forsample collection or waste. FIG. 4A provides an embodiment comprising asample collection outlet, a sample waste outlet, and a carrier wasteoutlet. The outlets are connected to dispensing nozzles or dispensingholes 314 in the faceplate through fluid vias. Dispensing nozzlesinterface with the baseplate.

The sorting region may also comprise one or more intersections thatconnect one or more additional inlets to the sorting region. Theseadditional inlets may be connected to the control reservoirs in thebody.

In preferred embodiments, the sorting region comprises at least amicrofluidic channel originating from the sample microfluidic channel, atrigger that can direct the flow of solution from the microfluidicchannel originating from the sample microfluidic channel into aselected, carrier microfluidic channel.

In some embodiments, the trigger is provided at an intersection betweenthe microfluidic channel originating from the sample microfluidicchannel and a first microfluidic channel providing liquid from a controlreservoir. FIGS. 4A-4B show one of the embodiments, where theintersection is connected to a trigger inlet by a microfluidic channel.Flow from the control reservoir, through the trigger inlet, and to theintersection provides the trigger flow.

When no flow or a low initial flow of liquid from the trigger inlet isprovided, the sample liquid flows into a default branch of theintersection's branch point. In preferred embodiments the default branchleads to a sample waste collection. When a flow or an increased flow isprovided from the trigger, the flow directs the sample liquid into aselected branch of the branch point.

The selected branch of the first branch point intersects at a secondintersection with a carrier microfluidic channel. The carriermicrofluidic channel is provided flow through a carrier inlet from acontrol reservoir. A plurality of microfluidic channels exit the secondintersection. In a preferred embodiment, two microfluidic channels, adefault branch channel and a selected branch channel, exit the secondintersection. In preferred embodiments, the default branch leads to acarrier waste collection and the selected branch leads to a samplecollection outlet. The carrier waste collection can be externallyvalved. The valve can be attached to the baseplate and connects thecarrier waste to the baseplate through a channel in the interface plate.In some embodiments, when the valve is open, a majority of the carrierfluid enters the carrier waste branch and preferentially exits throughthe carrier waste. When the valve is closed, a majority of the carrierfluid enters the sample collection branch and preferentially flows intothe sample collection outlet. The open/closed state of the valve isdependent upon the signal, signal change, lack of signal, or lack ofsignal change detected in the sensing region. In some embodiments, thevalve control is automated.

The microfluidic nature of the exit channel requires significantly lessfluid to advance sorted particles into an exit droplet than sorters withlarger dimensions. In some embodiments, the total exit droplet volume isless than 10 microliters, 10 microliters to 15 microliters, or greaterthan 15 microliters. In preferred embodiments, the fluid volume used toadvance sorted particles is less than or about 0.1 microliters in themicrofluidic chip and less than or about 5 microliters within theinterface plate channels. The reduction in required fluid increases thefrequency of switching the branch as a given aliquot progresses throughthe microfluidic channels while ensuring that the aliquot comprises asingle particle. This increase in switching frequency decreases the timerequired for a target particle to exit the cartridge as an isolate,thereby increasing the overall throughput.

FIGS. 6A-6G show an assembled microfluidic chip 620 and microfluidicchip mold 618, with detection and sorting regions. In the depictedembodiment, the detection or sensing region has both optical sensing(e.g., fluorescent sensing) and electrical sensing capability. Afluorescent sensing embodiment is shown in FIG. 15.

In some embodiments, optical sensing occurs prior to the particleentering an electrical detection or sensing location. In someembodiments, optical sensing occurs after the particle has exited froman electrical sensing or detection location. In embodiments where theelectrical sensing is used to trigger an LED or other light source,optical detection occurs after electrical sensing.

In some embodiments, the microfluidic chip comprises a sensing regioncomprising one or more nodes proximal to a conduit, as depicted in FIGS.5A-6G. In some embodiments, the microfluidic chip comprises a pluralityof nodes proximal to one or more conduits. For example, FIGS. 5A-6Gdepict an embodiment of a microfluidic chip comprising a sensing regioncomprising a conduit proximal a node on either side. In someembodiments, a microfluidic chip comprises a sensing region with nonodes. In some embodiments, a microfluidic chip comprises a sensingregion or sensing regions comprising a plurality of nodes, includingmore than two, more than three, more than four, or more than five nodes.In some embodiments, one or more of the nodes comprise functionalizedsurfaces. In some embodiments, none of the nodes comprise functionalizedsurfaces.

The volumes of the microfluidic channels can be described ascylindrical, prismatic, conical, or any other geometry or anycombination of geometries. As depicted in the figures (by way of exampleonly), the microfluidic channels are rectangular prisms. The effectivediameter of the channel connecting the sample inlet and the sensingregion in the figures is 0.35 mm, the effective diameter of the channelentering and exiting the sensing region where a particle is electricallydetected and characterized is 0.035 mm, and the effective diameter ofthe node within the sensing region where a particle is electricallydetected and characterized is 0.105 mm. In the depicted exemplaryembodiment, the distance from the sample inlet to the sensing regionabout 3.4 mm, the length of the sensing region is about 0.105 mm, andthe length of the channel connecting the sensing region to the sortingregion is about 1.7 mm. It should be understood that these dimensionsare meant as examples only and are not limiting.

FIGS. 7A-8I are an exemplary cartridge assembly, including a combinationof components described above with reference to FIGS. 2A-6G.

FIGS. 9A-9N are an exemplary base plate of an associated hardwareplatform. The base plate 924 includes mounting locations (or “mounts”)on its underside that are configured to interface with a hardwareplatform and/or electronics board. In some embodiments, the cartridgeassembly docks in a cartridge alignment cradle 926 of the base plate,thereby connecting it to access valves through liquid vias. As shown inFIGS. 9A-B and 9E-9F, the cartridge alignment cradle 926 can comprise aclear (unobstructed) location for collecting a sample.

FIGS. 10A-10B depict an exemplary cartridge 1000 mated with an exemplarybase plate 1024 of a hardware platform. FIG. 10A depicts a cartridge1000 that has been placed into the base plate 1024 of the hardwareplatform (the remainder of the hardware platform is not shown).

FIG. 10B shows exemplary locations 1026 of valve manifolds in the baseplate 1024.

The cartridge trigger reservoir can be connected to a valved port in thebase plate 1024 of the hardware platform, and carrier waste is routed toa valved port in the base plate 1024. In some embodiments, connectionsare made using o-ring sealing. FIG. 10B depicts an embodiment where thetrigger reservoir of the cartridge 1000 is connected to the base plate1024 using o-ring sealing.

FIGS. 11A-11L show an exemplary lid 1126 with a sealing surface. In someembodiments, the lid is separate from the cartridge and serves as a topplate that is positioned atop the cartridge assembly. In otherembodiments, the lid 1126 is part of the cartridge. In some embodiments,the lid is disposable. In the depicted embodiment of FIGS. 11A-11F, thisis accomplished using linear bearing ports 1128A and 1128B, which passthrough the lid 1126 at sites near the lid's edges.

Pressure applied to the chambers in the cartridge may be controlledusing the pressure access ports that traverse the lid.

The top of the lid 1126 of FIGS. 11A-11F defines a light filter cuberecess 1130 for positioning an optic system. When positioned, the opticsystem is aligned with a light access and filtration core within thelight filter cube recess 1130 that traverses the lid. Through thisaccess core, the optic system can access the light pipe or light borerunning through the cartridge (see, e.g., FIG. 15).

FIGS. 12A-12L depict an exemplary pressure regulator manifold. Thepressure regulator manifold 1232 bolts onto the hardware platform todrive the pressure inputs into the cartridge. The manifold can beattached to the hardware platform in any manner that allows for theapplication of air pressure to the cartridge cavities through themanifold's regulators. In a preferred embodiment, when air pressure isapplied through the pressure regulator manifold's regulators, the topplate of the instrument or a lid covers the cartridge cavities.

The pressure regulator manifold 1232 depicted in FIGS. 12A-12L isconfigured to provide 3-way valve access for solution flow. It furthercomprises a main pressure inlet on its side and regulated pressure outports on its top to control the pressure-driven flow of the solutions inthe cartridge's chambers.

FIG. 13 depicts an exemplary subsystem 1334 layout. FIG. 13 shows theassembly of a top plate 1326 (e.g., as described above with reference toFIGS. 11A-11L), a cartridge bearing a microfluidic chip substrate andmold 1336 (e.g., including a cartridge body as described above withreference to FIGS. 2A-2I, and a microfluidic chip substrate and mold asdescribed above with reference to FIGS. 4A-6G), a base plate 1324 (e.g.,as described above with reference to FIGS. 9A-9N) with electricalsensing contacts 1340, and a collection module 1344, shown in FIG. 13 tocomprise a 96-well plate output container 1342. Other elements of thesubsystem are not shown.

FIGS. 14A-14C are a schematic representation of the fluid paths in theexemplary subsystem depicted in FIG. 13. In the depicted embodiment,when a new cartridge is used, the solution sample fluid path does notinteract with any surfaces that were previously contacted with samplefluid loaded into an earlier cartridge.

FIG. 14A shows a trigger line fluid path running from trigger cavity inthe cartridge to the microchip trigger inlet. In this embodiment, theflow of solution is controlled by a valve indicated as an “X” in FIG.14A.

FIG. 14C shows a carrier line fluid path of the solution loaded into thecarrier cavity in the cartridge. Dependent on the flow of fluid in thecarrier line and whether the valve (depicted as an “X” in FIG. 14C) isopen or closed, the carrier fluid will either preferentially flow to theupper, sample collection path or the lower, waste path. In preferredembodiments the fluid in the carrier line is directed to the collectionor waste path in response to a signal or signals detected as a particleis in the sensing region or regions of the microfluidic chip.

FIG. 14B shows a fluid path of the solution in the sample cavity of thecartridge. Dependent on the flow of the fluid from the sample line andthe trigger line, the sample solution will flow to the upper, waste pathor to the lower sample, collection path. In preferred embodiments, thedirection of the sample solution is determined in response to a signalor signals detected as a particle is in the sensing region or regions ofthe microfluidic chip.

FIG. 15 is an embodiment of an LED-based fluorescent detection system1546. The grey rectangles depict the cartridge 1500. The central column1548 depicts the light pipe or light bore (“multimode fiber” or “freespace”, according to some embodiments) that runs through the verticallength of the cartridge 1500 and is positioned above the sensing regionof a microfluidic channel of a microfluidic chip 1520. Above the lightpipe 1548 is a dichroic mirror 1558 and fluorescent emission and/ordetection assembly 1550 that includes a quantitative fluorescenceremote-access detector (photodiode 1556 with fluorescent filter 1560 insome embodiments). A light source (LED in some embodiments) surfaceemitter 1552 is filtered by excitation filter 1554 to isolate theexcitation wavelength (e.g., blue, however other wavelengths can beused).

FIGS. 16A-16JD depict an example of an overall system assembly 1662.

FIG. 17 shows an exemplary scheme 1764 for detection and/or sensing andisolation. The arrow in the “carrier” row depicts the flow direction ofa solution that will carry a sorted particle of interest to a collectiondevice. This carrier row depicts a microfluidic channel that is withinthe microfluidic chip (see, e.g., FIGS. 6A-6G). In some embodiments, theparticle-bearing solution is the sole means by which the particle(s) ofinterest are carried (i.e., no sheath or other additional liquid isused).

A cartridge is designed to align its microfluidic architecture with thesensing/detection module and the collection module of the hardwareplatform.

The arrow in the “sample” row depicts the flow direction of a solutionthat carries particles from the sample reservoir of the cartridge. Thissample row depicts a microfluidic channel that is within themicrofluidic chip stage of the cartridge. Sample particles pass throughthe “sensing region” one-by-one. Each particle is interrogated in thesensing region, preferably by a combination of electrical andfluorescent detection elements. Combined electrical and fluorescentsignals from the particle are analyzed and the particle is identified.If the particle is identified to be a particle of interest, a trigger isactivated that directs the flow of solution into the carriermicrofluidic channel such that the particle of interest can becollected/isolated. In some embodiments, the trigger directs the samplesolution by altering the solution flow. In some embodiments, the triggerintroduces a crossflow of buffer or liquid to direct the samplesolution.

Combined with unique device geometries in the sensing region, signalprocessing algorithms calculate the electrical and/or fluorescentsignals from the particle resulting in a high sensitivity of detectionand dynamic range while tracking every particle. Active isolationtriggers and collects each particle of interest in a manner thatmaintains particle integrity, e.g. cell viability.

Functional Capabilities

No prior live particle isolation system can be used to provide all ofthe following features of the instant technology: recovery, single-cellisolation efficiency, capability of using a heterogeneous sample input,purity, the preservation of particle integrity, capability of using abroad sample input volume, speed of function, contamination-freeoperation, and degree of automation.

The instant technology allows for separately or individually detectingand characterizing any or all particles in a sample. After a particle isdetected or characterized, it can be directed/routed for collection orremoval from the sample output. In some embodiments, optical andelectrical detection and characterization of individual particles insolution allows for the recovery of rare particles or cells in solution.In some embodiments, the rare particles may be diluted in solution. Insome embodiments, rare particles are not diluted in solution. In someembodiments, the solution may comprise rare particles as well as commonparticles in greater abundance. In some embodiments, the rare particlesmay be cancer cells, circulating cancer cells, blood cells, white bloodcells, stem cells, transformed or transfected cells, or other cells orcell types. In some embodiments, the rare particles may be cells thatoverexpress one or more cell surface proteins. In some embodiments, therare particles may be cells that overexpress one or more intracellularproteins. In some embodiments, the rare particles may be characterizedby size. In some embodiments, the rare particles may be characterized bya combination of size and protein marker profile. In some embodiments,the particles are detected and not characterized. In some embodiments,the particles or cells may be at low concentrations or physiologicallyrelevant concentrations. In some embodiments, the system can operatewith only optical detection or only electrical detection.

Embodiments described herein allow for efficient single-cell isolation.In a preferred embodiment, combined electrical and optical detectionalgorithms control microfluidics for highly accurate single cell sortingof a desired cell phenotype from heterogeneous samples.

The efficiency of single-cell isolation can be defined as the number ofdesired (and, optionally, viable) single cells that are successfullydeposited into individual wells in a microtiter plate (e.g., a 96-wellplate), or any other receptacle. For example, 90 wells on a 96-wellplate having single cells would result in a “yield” or “isolationefficiency” of 93.75%. In some embodiments, combined electrical andoptical detection results in an isolation efficiency of greater than90%, greater than 91%, greater than 92%, greater than 93%, greater than94%, greater than 95%, greater than 96%, greater than 97%, greater than98%, or greater than 99%.

The input sample can be a solution or mixture comprising a homogenous orheterogeneous population of particles or cells. If the sample isheterogeneous, the particles of interest may be a majority or aplurality or a minority of the particles in the mixture. In someembodiments, the particles of interest are a substantial majority of theparticles in the mixture. In some embodiments, the particles of interestare about 50% of the particles in the mixture. In some preferredembodiments, the particles of interest are a minority of the particlesin the mixture. The ratio of particles of interest to total particles inthe mixture can be less than 1:1, less than 1:10, less than 1:100, lessthan 1:1000, about 1:10⁶, about 1:10⁷, about 1:10⁸, about 1:10⁹, about1:10¹⁰, about 1:10¹¹, about 1:10¹², or less than 1:10¹². For example,cancer cells or circulating tumor cells can be detected from a bloodsample where the target cells are present on the order of one targetcell per millions of background (non-target) cells.

Sorting and isolating uses of the instant technology result in a highpurity output. In one embodiment of dual detection, combined electrical(NPS-based) and LED-based fluorescent detection reduces detection offalse positives. Depending upon the implementation, using systems ormethods described herein can result in a reduction of false positivessuch that false positives constitute less than 10%, less than 9%, lessthan 8%, less than 7%, less than 6%, less than 5%, less than 4%, lessthan 3%, less than 2%, or less than 1% of the total isolatedcells-of-interest. In some embodiments, an LED-based excitation sourceor sources are used. In some embodiments, a laser-based excitationsource or sources are used. In embodiments where single particles areisolated in buffer, this high purity results in greater than 90%,greater than 91%, greater than 92%, greater than 93%, greater than 94%,greater than 95%, greater than 96%, greater than 97%, greater than 98%,or greater than 99% of the receptacles used for output collectioncontaining a single target particle. In embodiments where targetparticles are sorted, this high purity results in the target particlesbeing greater than 90%, greater than 91%, greater than 92%, greater than93%, greater than 94%, greater than 95%, greater than 96%, greater than97%, greater than 98%, or greater than 99% of all sorted particles in anoutput sample or samples.

Due to the capability of the system to have “bench-top” dimensions, thesystem can be placed in a small sterile environment such as a fume hoodor a culture hood. This further ensures the purity and/orcontamination-free nature of the sample.

The preservation of particle integrity and single-particle isolationenables additional downstream confirmation and characterization of theparticles. Viability of cells can be validated, for example, usinglive/dead assays such as Trypan Blue or Propidium Iodide dyes andmicroscopy, or CellTiterTM, or by culturing. In embodiments where theparticles are viable cells, the technology enables founding homogenous,clonal colonies from a single-cell isolate. Phenotype calling with suchcolonies ensures calling uniformity and reproducibility. Phenotypingtechniques are known in the art and include PCR, Sanger sequencing,next-generation sequencing, antibody-based staining, and microscopy. Insome embodiments, the phenotyping of sorted cells is used to confirmproper isolation and sorting. In some embodiments, phenotyping is usedto characterize cellular properties that founder isolates were notoriginally sorted by.

No prior incubation or binding steps are necessary for single-particledetection and isolation. In some embodiments, the particle comprises afluorescent reporter tag. In some embodiments, the particle may be acell or transfected cell comprising the fluorescent reporter tag. Insome embodiments, the fluorescent reporter tag is an expressedfluorescent protein such as green fluorescent protein (GFP). In someembodiments, a particle comprising a fluorescent reporter tag may allowfor staining-free optical detection. In some embodiments, the excitationsource (e.g. an LED) is coupled to the electrical detection of aparticle or cell, such that the excitation source is selectivelyactivated dependent upon the electrical detection and/orcharacterization profile of a particle or cell. Photobleaching andcytotoxic events are reduced by limiting exposure to an excitationsource through this coupling, leading to increased cell viabilitycompared to traditional cell detection, characterization, and sortingtechniques.

The present technology permits sample volumes from 1 microliter togreater than 10 milliliters to be used, thereby removing the need forconcentration or dilution steps. FIGS. 2G-2I depict an exemplary samplereservoir with a maximum sample volume of about 10 milliliters. However,the maximum sample volume can be any volume that the sample reservoircan hold, and there are no limits to the size of the sample reservoir.In some embodiments, the sample reservoir may contain more than 10milliliters, 15 milliliters, 20 milliliters, 30 milliliters, 50milliliters, 100 milliliters, or 1 liter. The sample volume can also beless than 10 milliliters. In some embodiments, the sample volume can beless than 10 milliliters, less than 5 milliliters, less than 1milliliter, less than 100 microliters, less than 50 microliters, lessthan 25 microliters, about 10 microliters, about 1 microliter, or lessthan 1 microliter.

The sorting of the instant technology can detect, characterize, and sortor isolate particles at high speeds. The rate can be altered byadjusting the concentration of particles in the sample and the flow rateof particles through the microchip. In some embodiments, particles ofinterest in a heterogeneous population can be isolated at rates of aboutless than 1 particle per minute, 1 particle per minute, 10 particles perminute, 100 particles per minute, 1000 particles per minute, about10,000 particles per minute, about 100,000 particles per minute, orgreater. In some embodiments, particles in a heterogeneous sample can besorted (i.e. target particles are collected separately from non-targetparticles) at rates of about less than 1 particle per minute, 1 particleper minute, 100 particles per minute, 1,000 particles per minute, 10,000particles per minute, 100,000 particles per minute, 1 million particlesper minute, 10 million particles per minute, 100 million particles perminute, 1 billion particles per minute, or greater. In some embodiments,particles of interest in a homogeneous population can be isolated atrates of less than 1 particle per minute, 1 particle per minute, 10particles per minute, 100 particles per minute, 1000 particles perminute, about 10,000 particles per minute, about 100,000 particles perminute, or greater.

The present technology is capable of complete automation after sampleloading, buffer loading, and software parameter input is provided to thesystem. This allows for high-speed, high-efficiency, automateddetecting, characterizing, sorting and isolating particles of interest.

System Operation and Workflow for Particle Isolation

Sample and Buffer Loading into Cartridge

A liquid sample containing the cell(s) or particles of interest areintroduced or loaded into the cartridge. In the exemplary cartridgedepicted in FIGS. 2A-2I, the liquid sample is introduced into the samplereservoir compartment.

The liquid sample can have a volume as small as 1 microliter. Themaximum volume of the liquid sample is limited only by the overall sizeof the cartridge, which is designed relative to the overall size of thehardware platform. In one embodiment, the maximum volume of the liquidsample is 10 milliliters. The liquid sample can have a target particleconcentration of 0.1 particle per milliliter to 10,000,000 particles permilliliter.

In one embodiment of the cartridge, the sample reservoir can hold up to10 mL or more of solution (see, e.g., FIGS. 2A-2I for a picture of thesample reservoir).

Running buffer solutions are also added to the cartridge. In oneembodiment of the cartridge, there are two “control reservoirs” intowhich running buffer solutions are added. For example, one such controlreservoir can provide a carrier flow while the other can provide atrigger flow, as discussed above. The running buffer solutions can becell culture media, phosphate buffer saline, oil, or any solutiondesired for output.

Cartridge Placed into Base Plate of Hardware Platform

By placing the cartridge into the base plate of the hardware platform, aclamping mechanism mates the cartridge to electrical, pneumatic,fluorescent, collection, and waste subsystems of the hardware platform(see, e.g., FIGS. 16A-16D.) The vertical alignment of the valve manifoldlocations between the cartridge and the hardware platform can benefitthe control of the flow or dispensing rate.

Operation Commencement

The pneumatic subsystem is activated in a manner that first causes anair pressure change in the sealed cartridge. The air pressure changecauses solution to flow from the sample reservoir through fluid channelsof the interface plate to the microfluidic channel in the microfluidicchip of the cartridge. Solution flow from the sample reservoir causesparticles to travel into the microfluidic channel. Pressurized air issupplied to the reservoirs through pressure regulators. The pressure canbe controlled, supplied, and tuned individually. In some embodiments,the pressure supplied to one reservoir is adjusted in response to itseffect on the flow from a different reservoir. In some embodiments, eachreservoir has an independently controlled regulator.

Sensing and Detection

As the particle-laden sample solution flows through the microfluidicchannel, the solution passes through one or more sensing regions of themicrofluidic channel. In some embodiments, the sensing region has asmaller diameter or width than non-sensing regions of the microfluidicchannel. The sensing region can include an orifice having a shape thatis spherical, square, rectangular, a polygon, or a combination ofoverlapping edges of these, or any other possible cross-section that islarge enough for particles to traverse. Using electrical (e.g., ACand/or DC current) and/or optical detection (e.g., single wavelength ormulti-wavelength), the presence of one or more particles of interest canbe detected. In preferred embodiments, electrical detection and/orcharacterization is conducted with direct current. In some embodiments,alternating current is used. In some embodiments, alternating and directcurrent are used in combination, either in serial or in parallel. Insome embodiments, optical detection is done with multiple wavelengths.In these embodiments, the technology may comprise multiple excitationsources, filter sets, and detectors. In some embodiments, the electricaldetection comprises producing a direct current flow across a region ofinterest within the microfluidic channel. In some such embodiments, RFcurrent is not simultaneously applied.

Numerous microchannel designs are suitable for detection. In someembodiments, microchannels comprise straight rectangular prism channels,cylindrical channels, or some other geometry channels. Microchannels cancomprise zero, one, two, or more than two nodes. Nodes in microchannelscomprising nodes can be of any appropriate geometry, for examplestraight rectangular nodes, cylindrical nodes, or some other geometry.In some embodiments, electrical and optical sensing are both used. Insome embodiments, optical sensing using an LED or laser source isfollowed by electrical sensing. In some embodiments, electrical sensingis followed by optical sensing with an LED or laser source. In someembodiments, electrical and optical sensing are performedsimultaneously. In some embodiments, only electrical or optical sensingis performed in a sensing region.

Isolation and Sorting

Upon detection of the one or more particles of interest, pressure isincreased on a trigger flow inlet to the channel, causing the one ormore detected particles of interest to be diverted into a second,“carrier” channel (see, e.g., FIG. 17). In some embodiments, the carrierchannel has no flow unless a particle has been directed into it. Inother embodiments, a base level flow is always provided to the carrierchannel. In some embodiments, the flow rate is altered after beingtriggered by detection and/or characterization of a particle in thesensing region.

Isolating droplets form as a result of liquid entering and exiting adispensing nozzle. Drop size can be modulated by physical properties ofthe exit channel including nozzle design, nozzle inner orifice diameter,nozzle outer orifice diameter, contact angle, surface coating,hydrophobicity of the channel, and applied vibration. Droplets can rangefrom sub microliter to several microliters (e.g. 50) in common usecases. It is envisioned that isolation droplets could be on themilliliter scale.

Solution that has been sorted is directed to an outlet in themicrofluidic chip mold that is connected to a nozzle in the faceplatethrough a fluid via and fluid channel. In some embodiments, this is doneby blocking the default branch of the branch point involving the finalcarrier microfluidic channel. The increased fluid flow to the selectedoutlet enables the formation of a droplet containing a single particleat the collection nozzle on the bottom of the interface plate. Thedroplet may dissociate from the nozzle by gravity, the force of the flowfrom the selection outlet, or a combination of the two.

The droplet from the nozzle may be directed into any desired collectionreceptacle. In some embodiments, each individual droplet is depositedinto a separate receptacle. In other embodiments, a plurality ofdroplets are deposited into the same receptacle. In certain embodiments,individual droplets are isolated by depositing each into a separate wellof one or more microtiter plates or any other appropriate receptacle.Receptacle plates may have any number of wells including 8, 16, 64, 96,128, 256, 384, or more than 384 wells. In certain other embodiments, aplurality of droplets, each containing an individual particle, aredeposited into the same receptacle and form a solution containing aplurality of particles. In these embodiments, the concentration and/orpurity of the selected particles may be increased relative to that ofthe sample solution loaded into the sample reservoir. In someembodiments, a droplet can contain more than one target particle.

Each droplet can contain different types of particles of interest.Systems described herein can include the ability to detect and activelysort multiple cell phenotypes.

The microfluidic nature of the technology allows for the volume of asingle collection droplet to be decreased. Minimizing the volume of thecollection droplets increases the probability that a given dropletcontains exactly one isolated particle. Carrier channels on themicrofluidic scale also decrease the time between detecting and/orcharacterizing a particle and its collection, thereby increasing therate of detecting, characterizing, and/or collecting particles in thesample solution.

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent.

Example 1 Sorting of Circulating Tumor Cells from a Blood Sample

FIGS. 18A-18C show cell sorting of circulating tumor cells from a bloodsample. Detection of breast cancer cell line cells, MCF-7 expressinggreen fluorescent protein (GFP), in a whole blood sample was performedusing electrical sensing. Detected MCF-7 cells were sorted away fromwaste. Breast cancer cell line cells, MCF-7 expressing green fluorescentprotein (GFP) were spiked into whole blood at physiologically relevantconcentration (near circulating tumor cell concentration) for a totalcellular sample concentration of about 10-1000 cells per milliliter. Thewhole blood/MCF-7-GFP solution was loaded into the sample reservoir. Aflow rate of 100 microliters/min was applied to the sample. Cells werecharacterized by electrical impedance in the microfluidic sensingregion. Cells possessing MCF-7-GFP impedance signatures were separatedfrom those that did not exhibit those signatures and collected (FIG.18A). The final concentration of the detected and collected MCF-7-GFPcells was roughly 100% of the MCF-7-GFP cells in the initial sample.FIG. 18A shows changes in the current in the microfluidic conduit overtime as the sample flowed through the microfluidic chip.

Traditional flow cytometry was performed separately on the collectedMCF-7-GFP and the waste to confirm correct sorting (FIG. 18B). Forwardscattering and side scattering measurements of the MCF-7-GFP collectiondisplayed a number large cells (high forward scattering) with highgranularity (high side scattering) indicating cancer cells. In contrast,the Waste collection lacked any large, highly granular cancer cells.

Further confirmation of correct sorting was performed by measuring greenfluorescence using traditional fluorescent flow cytometry (FIG. 18C).The collection output contained a number of cells that fluoresced at theGFP wavelength confirming high accuracy sorting by the instanttechnology. 98% of the MCF-7-GFP cells were correctly identified andrecovered from the spiked whole blood sample.

Example 2 Isolation of a Cancer Cell from a Cell Culture Sample

An MCF-7 cell culture was loaded into the sample reservoir at aconcentration of 5000 cells per milliliter. A flow rate of 250 μL/minwas applied to the sample. Cells were detected by electrical sensing inthe microfluidic sensing region. Individual MCF-7 cells were isolatedfrom a sample comprising a plurality of cells and dispensed into singlewells of a 96-well plate. Roughly 94% of the wells contained a singlecell. The remaining 6% of wells contained no cells or multiple cells.FIGS. 19A-19C show three example single collection wells containing asingle, isolated MCF-7 cell.

Example 3 Isolation of a Cancer Cell Using Fluorescence Based Selection

Filtered MCF7-GFP cells (Cell Biolabs cultured in Dulbecco's ModifiedEagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS)) were loaded intothe sample reservoir at a concentration of 10,000 cells/mL. A flow rateof 100 μL/min was applied, driven by a pressurized air supply. Thetrigger and carrier fluids were 1× Phosphate Buffered Saline. Themicrofluidic chip of the system comprised channels fabricated out ofcyclic olefin copolymer with a single node in the sensing region.Fluorescent detection of single cells was performed using a 473 nmexcitation laser and a photodetector. Single cells were dispensed into a96-well plate housed on a moving X-Y stage. The X-Y stage shifted to anew well after each detected cell was dispensed. FIGS. 22A-C showfluorescent images of three examples of single collection wellscontaining a single, fluorescently isolated MCF7-GFP cells.

Example 4 Fluidic Sorting is Independent of Particle Size

FIG. 21 depicts MCF7 cell clusters isolated using the platform.Unfiltered MCF7-GFP cells cultured in DMEM with 10% FBS was loaded intothe sample reservoir at a concentration of 10,000 cells/mL. A flow rateof 100 μL/min was applied, driven by a pressurized air supply. 1×Phosphate Buffered Saline was used as the trigger and carrier fluids.The microfluidic chip of the system comprised channels fabricated out ofcyclic olefin copolymer with a single node in the sensing region.Node-pore sensing was used to selectively gate for larger cell clustersof about 35 microns or larger. A four-point terminal measurement of thecurrent across the node-pores was performed. As cell clusters wereidentified, trigger fluid was pulsed to allow for the selectivecollection and isolation of the target clusters.

Example 5 Cells Isolated from a Cell Culture Sample Maintain TheirViability

MCF7-GFP, BC3 (ATCC CRL-2277), or Jurkat Clone E6-1 (ATCC TIB-152) cellswere cultured in DMEM with 10% FBS, Roswell Park Memorial Institute(RPMI) 1640 with 20% FBS, or RPMI 1640 with 10% FBS, respectively.MCF7-GFP, BC3, or Jurkat Clone E6-1 samples were loaded into the samplereservoir along with their respective medium at a concentration of500,000 cells per milliliter. Trigger and carrier fluids were 1×Phosphate Buffered Saline. A flow rate of 100 μL/min driven by apressurized air supply was applied to the samples. The microfluidic chipof the system comprised channels fabricated out of cyclic olefincopolymer with a single node in the sensing region. Node-Pore Sensingpulses were used for detection and configured to pulse the trigger fluidand allow for isolation of 8-25 microns. As single cells transitedthrough the device, a four-point terminal measurement of the currentacross the node-pores was performed.

100 μL of collected cell volumes were stained with 100 μL of TryptanBlue to assess viability for each of the cell types. MCF7-GFP, BC3, andJurkat Clone E6-1 cells that had not been run through the detection andisolation scheme were taken from the original input sample. Thesecontrol cell samples were also stained with Tryptan Blue. FIG. 20depicts the percent viability for both the isolated and control cellsamples as analyzed using microscopy.

Example 6 Isolation of Colloids

Colloids of 5 μm, 10 μm, 15 μm, and 20 μm diameters have been detectedand isolated.

Example 7 Exemplary Operation

Fill cartridge chambers with respective liquids (e.g., 8 mL of 1×phosphate buffered saline in carrier and trigger fluid chambers, 10,000MCF7 cells/mL media in sample chamber).

Place cartridge onto instrument baseplate—see valve manifold locationsand interface plate design (FIGS. 10A-10B). Auto-alignment ensures 1)positioning of carrier waste routing on interface plate into hole inbase plate routing to external microvalve and then back to interfaceplate toward carrier waste outlet, 2) trigger hole on interface platerouting into hole in base plate routing to external microvalve and thenback to interface plate toward trigger inlet, 3) collection/dispensingnozzle positioned over desired collection chamber for drop collection,4) electrical contact between electrode pads on chip and systemelectrical contacts, 5) positioning of light bore under LED in topplate.

Clamp cartridge using instrument. This connects pressurized air sources(turned off until beginning of test) with the cartridge chambers.

Pressure can be tuned/retuned using knobs for each of the chambers untildesirable fluid switching is achieved (i.e., when external trigger valveis open, sample fluid flows into collection outlet).

Valve on-times, off-times, and dead-times can be set for a desiredsetting (e.g., when a particle of interest is detected, turn on theexternal trigger microvalve for 100 milliseconds (this is default off)and close the external carrier microvalve for 5 seconds (this is defaultopen to ensure the majority of carrier fluid is routed to waste and notthe collection droplet when there is no detection, and closing theexternal microvalve ensures that carrier flow only goes through thecollection nozzle, thus accelerating the drop creation and detachmentonce a particle has been detected). The carrier microvalve close time iscalibrated with droplet detachment time. The trigger microvalve has adead time associated with this droplet time as well, i.e., the triggermicrovalve will not trigger again until the drop is detached to ensurethe drop contains only one target particle if single-particle dispensingis desired. If single-particle per well dispensing is desired, stagemovement is calibrated with the droplet timing (i.e., move to next wellchamber after waiting for detection of single particle and droplet todetach and fall into the well).

Electrical sensing offset voltages for sensing can be set as desired(larger magnitude for larger pulses, intermediate magnitudes forselection as “gates”). Fluorescent settings can likewise be calibrated(e.g., LED voltage, photodectector/PMT gain, etc.). Thresholds can thenbe set for proper gating as desired.

Pressurized air is turned on and the test runs to completion.

The collection plate/tube can be removed from the instrument withparticles sorted/dispensed.

Example 8 Exemplary System

An exemplary system includes the disposable cartridge shown in FIGS.2A-2I, the interface plate shown in FIGS. 3A-3F, and the microfluidicchip shown in FIGS. 4A-6G.

The disposable cartridge body has a sample reservoir, a carrierreservoir, and a trigger reservoir. The sample reservoir has a maximumvolume of 10 milliliters. Sample containing particles of interest areloaded into the sample reservoir at a volume of 1 microliter or more upto the sample reservoir's maximum volume. The carrier reservoir and thetrigger reservoir each have a maximum volume of 5 milliliters. Carrierand trigger fluids are loaded into the carrier and trigger reservoirseach has a volume of 100 microliters or more up to the respectivereservoir's maximum volume.

The bottom of the sample reservoir, carrier reservoir, and triggerreservoir each have a fluid inlet aperture of 1/32 inches in diameter.These apertures provide access for fluid in each reservoir to theinterface plate attached to the disposable cartridge in the system. Thefloor of the sample reservoir is sloped such that sample fluid loadedinto the sample reservoir accumulates at or proximal to the samplereservoir's aperture.

A light pipe core is positioned in the wall separating the reservoirsand can be seen in FIGS. 2A-2C and FIGS. 2G-2I. The light pipe traversesthe cartridge body vertically without contacting the internal volumes ofthe reservoirs. The light pipe provides passage for emission andexcitation wavelengths through the cartridge body to and from thesystem's microfluidic chip.

The cartridge body has overall dimensions of 54 mm×46 mm×32.4 mm and ismachined out of acrylic.

The interface plate of the exemplary system attaches to the bottom ofthe disposable cartridge body. The bottom of the interface plateincludes dispensing nozzles for dispensing sample collection containingparticles of interest detected and/or characterized by the system,sample waste, and carrier waste (see, e.g., FIGS. 3A and 3E). The upperside of the interface plate provides channel ways connecting theapertures of the disposable cartridge body to the microfluidic chipinlets and from the microfluidic chip's outlets to the dispensingnozzles on the bottom of the interface plate (see, e.g., FIGS. 3C and3D). The upper side of the interface plate contains channel ways 312that connect the sample and control reservoirs of the cartridge bodythrough the reservoirs' apertures to the baseplate through holes on thebottom of the interface plate. Connection to the baseplate allows forexternal valving. The bottom of the interface plate also has bondingridges to ensure proper attachment of the interface plate with themicrofluidic chip (see, e.g., FIG. 3F). Through the center of theinterface plate is a hole that aligns with the light pipe in thecartridge body and allows light to traverse the interface plate to andfrom the microfluidic chip.

The interface plate has overall dimensions of 54 mm×46 mm×4.3 mm and ismachined out of polycarbonate. It is adhered to the bottom of thecartridge body via a pressure sensitive adhesive.

The microfluidic chip includes a microfluidic chip substrate and amicrofluidic chip mold (FIGS. 4A-6G). The microfluidic chip substratecomprises electrodes made of a copper adhesion layer with a platinum toplayer. The electrodes are provided electrical current and serve aselectrical contacts for sensing and detecting particles in the sensingregion. The microfluidic chip mold comprises microchannels whereparticles are sensed, detected, characterized, sorted, and/or isolated.

The microchannel geometry is shown in FIGS. 4A-4B. Fluid from the samplereservoir, provided passage through the interface plate, enters themicrofluidic chip through the sample inlet. The particles in the samplefluid are advanced through a microchannel with a 0.35 mm diameter to thesensing region. Inside the sensing region is a node with a diameter of0.105 mm. On either side of the node in the sensing region themicrochannel has a diameter of 0.035 mm. As a particle passes throughthe sensing region it is electrically detected and/or characterizedusing the microfluidic substrate's electrodes. The sensing region isalso aligned with the light pipe in the interface plate and thecartridge body, such that a particle passing through the sensing regionis exposed to an excitation wavelength, and emission wavelengths fromthe particle are detectable and/or characterizable.

The microfluidic channel exiting the sensing region has a diameter of0.35 mm and intersects with a microchannel from the trigger inlet. Thetrigger inlet is provided a flow of trigger fluid from the triggerreservoir through the interface plate. Dependent upon the sensing and/orcharacterization signal, a flow is provided from the trigger inlet todirect or route the particle in the first intersection. In the absenceof the trigger flow, the particle travels to the sample waste outlet andis dispensed through the connected output nozzle in the interface plate.Upon the application of the trigger flow, the particle is directed to amicrochannel that intersects with a microchannel from the carrier inlet.The carrier inlet is provided a flow of carrier fluid from the carrierreservoir through the interface plate. A valve controls the flow ofcarrier fluid into the carrier waste outlet. When the valve is open, thefluid in the second intersection is preferentially directed to thecarrier waste and is dispensed through the connected output nozzle inthe interface plate. When the valve is closed in response to the signalor signal change measured in the sensing region, the fluid and thedetected and/or characterized particle is directed to the samplecollection outlet. The sample collection outlet is connected to thethird nozzle in the interface plate where particles are dispensed forcollection.

Both the mold and the substrate are composed of plastic 1/32 inchesthick and are bonded together through a combination of heat, lamination,and solvent.

An assembly of the cartridge is shown in FIGS. 7A-8I and the assembly isshown in relation to an exemplary subsystem including an optical source,top plate, base plate, and collection module in FIG. 13.

What is claimed is:
 1. A particle sorter cartridge, comprising: amicrofluidic chip including a sensing or detector region; and a bodydefining a plurality of reservoirs therein; and optionally an interfaceplate configured to connect to the body and to the microfluidic chip;wherein in use, and upon detection of a particle in a particle flowwithin the sensing or detector region, a solution flow within themicrofluidic chip is diverted by application of a trigger flow to atleast one of: sort and isolate the detected particle from the particleflow.
 2. The cartridge of claim 1, wherein one or more of themicrofluidic chip, the cartridge body, and the optional interface plateare disposable.
 3. The cartridge of claim 1, wherein the plurality ofreservoirs includes a sample reservoir, a control fluid reservoir, and atrigger reservoir.
 4. The cartridge of claim 3, wherein the trigger flowcomprises fluid from the trigger reservoir.
 5. The cartridge of claim 1,further comprising an electrical detector that is operably coupled tothe detector region.
 6. The cartridge of claim 1, further comprising anoptical detector that is operably coupled to the detector region.
 7. Thecartridge of claim 5, further comprising an optical detector that isoperably coupled to the detector region.
 8. The cartridge of claim 5,wherein the detection of a particle in the detector region is based upona signal generated by the electrical detector.
 9. The cartridge of claim6, wherein the detection of a particle in the detector region is basedupon a signal generated by the optical detector.
 10. The cartridge ofclaim 7, wherein the detection of a particle in the detector region isbased upon at least one of: a signal generated by the electricaldetector; and a signal generated by the optical detector.
 11. Thecartridge of claim 10, as depicted in FIG.
 7. 12. A particle sortingsystem, comprising: a base plate; and a cartridge assembly, the baseplate configured to receive and connect to the cartridge assembly; thecartridge assembly including: a microfluidic chip including a sensing ordetector region; and a body defining a plurality of reservoirs therein,the cartridge assembly configured such that, in use, and upon detectionof a particle in a particle flow within the detector region, a solutionflow within the microfluidic chip is diverted by application of atrigger flow to at least one of: sort and isolate the detected particlefrom the particle flow.
 13. The cartridge of claim 12, wherein one ormore of the microfluidic chip and the cartridge body are disposable. 14.The system of claim 12, wherein the plurality of reservoirs of the bodyof the cartridge assembly includes a sample reservoir, a control fluidreservoir, and a trigger reservoir.
 15. The system of claim 14 whereinthe trigger flow comprises fluid from the trigger reservoir.
 16. Thesystem of claim 15, wherein the trigger flow is gated by valving. 17.The system of claim 16, wherein the valving is external to the cartridgeassembly.
 18. The system of claim 14, wherein the cartridge assembly isfurther configured such that, in use, and upon application of a triggerflow, a solution flow within the microfluidic chip is diverted byapplication of a carrier flow to at least one of: sort and isolate thedetected particle from the particle flow.
 19. The system of claim 18,wherein the carrier flow comprises fluid from the control fluidreservoir.
 20. The system of claim 19, wherein the carrier flow is gatedby valving.
 21. The system of claim 20, wherein the valving is externalto the cartridge assembly.
 22. A particle sorting system as described inExample
 8. 23. A method for active isolation of a particle, the methodcomprising: loading a sample comprising a mixture of particles into thecartridge assembly of the system of any one claims 12-22, detecting aparticle of interest by optical and/or electrical signals generated bythe particle of interest as it passes through the sensing or detectorregion of the system of any one claims 12-22, actively sorting theparticle of interest based on the optical and/or electrical signalsthrough software means that causes the solution flow within themicrofluidic chip to divert by application of a trigger flow, depositinga droplet comprising the particle of interest into a collectionreceptacle.
 24. The method of claim 23, wherein the system is the systemof claim 22, and wherein the detecting step involves simultaneousoptical and electrical signals.
 25. The method of claim 24, wherein thedetecting step provides a method to measure the velocity of the particletraversing the sensing or detector region.
 26. The method of claim 25,further comprising a step of increasing the velocity of the particle ofinterest as it passes through the sensing or detecting region such thatthe total time of exposure to optical signals is less than 100milliseconds.
 27. A method for active isolation of a particle, themethod comprising: flowing a mixture of particles through a microchannelcomprising a sensing or detector region; detecting a particle ofinterest as it passes through a sensing or detector region; divertingthe flow of the particle solution by the application of another flowupon detection of a particle of interest in the detector or signalingregion, thereby actively sorting the particle; and depositing a dropletcomprising the particle of interest into a collection receptacle. 28.The method of claim 27, wherein detecting is by optical and/orelectrical signals generated by the particle of interest as it passesthrough the sensing or detector region of the microchannel.